-Etiologic Risk Group (see link below): Risk group 2
-Disease Information (sort of like the Intro to your Mini Research Write up): Toxoplasma gondii is a ubiquitous parasitic protozoan that causes the disease toxoplasmosis. Toxoplasmosis is a neurologic infection prevalent among people with acquired immunodeficiency syndrome or immunosuppressive cancer. However, humans with normal immunity can still contract the disease and certain symptoms, such as fever, malaise, and lymphadenopathy, will appear. Extreme cases include blindness, epilepsy, and even death [1]. T. gondii is estimated to infect half of the world's population, and it can virtually infect all warm-blooded animals [2]. Its economic impact is estimated to be $7.7 billion annually in the United States alone [3]. Infection among humans occurs mainly due to eating raw or uncooked meat, having contact with the parasite's oocysts via touching soil, or congenitally acquiring the disease through the placenta [1]. Currently, there is no licensed vaccine for humans [2], so much research still needs to be done.
-Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.):NCBI, ToxoDB
-Essentiality of this protein: Pyruvate kinase is an important metabolic regulator in T. gondii that catalyzes the final step in glycolysis by converting phosphoenolpyruvate into pyruvate and producing ATP from ADP.
Structure (PDB or Homology model) -PDB # or closest PDB entry if using homology model: 3EOE
-For Homology Model option: -Show pairwise alignment of your BLASTP search in NCBI against the PDB -Query Coverage: -Max % Identities: -% Positives -Chain used for homology:
-Expression Information (has it been expressed in bacterial cells): Pyruvate kinase has been expressed in BL21(DE3) competent E. coli cells.
-Purification Method: Metal ion affinity with the enzyme's 6x His tag (source)
-Image of protein (PyMol with features delineated and shown separately):
Figure 2: PyMol representation of pyruvate kinase (PDB: 3EOE) shown as cartoon. Each subunit is colored differently: chain A yellow, chain B green, chain C blue, and chain D orange.
-Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MHHHHHHSSGRENLYFQGIRMSQILEPRSEEDWTAHRTRIVCTMGPACWNVDTLVKMIDAGMNVCRLNFSHGDHETHART
-Length of your protein in Amino Acids: 511 amino acids
-Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 55.863 kDa
-Molar Extinction coefficient of your protein at 280 nm wavelength: 24410 L/mol.cm (assuming all Cys residues are reduced) and 25035 L/mol.cm (assuming all pairs of Cys residues form cystines)
Figure 3: TMpred output for the amino acid sequence of pyruvate kinase (PDB: 3EOE). The graph represents TMpred's prediction of membrane-spanning regions and their orientation.
-CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
ATGCACCACCATCACCATCATTCTTCTGGCCGCGAGAATTTATACTTCCAGGGTATTCGCATGTCTCAAATCCTGGAACCCCGTAGCGAAGAAGATTGGACCGCTCATCGCACACGCATCGTGTGTACGATGGG
ACCAGCGTGTTGGAACGTCGATACGCTGGTGAAAATGATTGACGCAGGTATGAATGTCTGCCGTTTAAATTTTTCACATGGTGATCACGAGACGCATGCGCGCACTGTGCAGAATATCCAAGAGGCTATGAAAC
AACGTCCCGAGGCCCGCCTTGCGATCTTATTGGATACGAAAGGGCCCGAAATCCGTACGGGGTTCCTGAAAGATCACAAACCAATCACCTTACAGCAAGGAGCAACGTTGAAAATTGTCACAGATTACAATTTA
ATTGGAGACGAGACAACCATCGCGTGTAGCTATGGGGCTTTGCCACAATCGGTTAAGCCGGGAAATACAATCTTAATTGCGGACGGCTCATTGAGCGTTAAGGTAGTGGAGGTAGGGTCCGATTATGTAATTAC
TCAAGCTCAAAACACTGCTACAATCGGAGAGCGTAAGAATATGAATCTGCCCAATGTTAAGGTACAACTGCCCGTCATTGGTGAAAAGGACAAGCACGACATTCTTAACTTTGGAATTCCAATGGGCTGTAATTT
TATTGCGGCAAGCTTCGTTCAGTCTGCTGACGACGTCCGTTACATCCGTGGACTGCTTGGTCCGCGTGGTCGCCATATCCGCATCATCCCCAAGATTGAAAATGTGGAAGGATTAGTGAACTTTGATGAGATCC
TTGCAGAGGCAGACGGCATTATGATCGCACGTGGTGACCTGGGGATGGAGATCCCCCCGGAGAAGGTCTTCTTGGCACAAAAAATGATGATCGCGAAATGTAATGTCGTGGGAAAGCCAGTCATCACGGCCA
CACAAATGTTGGAGTCCATGATCAAAAATCCCCGTCCGACTCGCGCGGAAGCGGCGGATGTAGCTAACGCGGTTCTGGATGGCACTGATTGCGTGATGCTGTCCGGCGAAACCGCAAACGGTGAGTTTCCT
GTCATCACTGTTGAGACTATGGCACGTATTTGTTATGAAGCGGAAACGTGTGTGGATTACCCTGCATTATACCGTGCGATGTGCTTAGCTGTACCCCCCCCGATCTCTACTCAAGAGGCGGTAGCTCGTGCCGC
AGTAGAGACGGCTGAATGCGTGAATGCCGCTATCATTTTGGCATTGACAGAAACGGGACAAACTGCCCGCTTAATCGCAAAGTACCGCCCCATGCAGCCCATTCTTGCCTTATCGGCTTCAGAGTCGACTATTA
AGCATCTTCAGGTAATTCGCGGAGTGACAACAATGCAGGTGCCTAGTTTCCAAGGTACGGATCACGTCATTCGTAACGCAATTGTTGTGGCTAAAGAGCGCGAGTTAGTAACAGAGGGCGAGTCCATTGTGGC
CGTTCACGGTATGAAAGAGGAAGTAGCGGGAAGCTCTAATTTATTGAAAGTTTTAACAGTAGAA
-GC% Content for gene (codon optimized): 49.1%
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
-References: 1. Jones, J.L.; Kruszon-Muran, D.; Wilson, M.; McQuillan, G.; Navin, T.; McAuley, J.B., Toxoplasma gondii Infection in the United States: Seroprevalence and Risk Factors. American Journal of Epidemiology2001, 154, (4), 357-365. 2. Liu, Q.; Das Singla, L.; Zhou, H., Vaccines against Toxoplasma gondii: Status, challenges, and future directions. Human Vaccines and Immunotherapeutics2012, 8, (9), 1305-1308. 3. Buzby, J.C.; Roberts, T., ERS updates US foodborne disease costs for seven pathogens. Food Review: The Magazine of Food1996, 19, 20-25.
-NCBI Gene # or RefSeq#: 7901097
-Protein ID (NP or XP #) or Wolbachia#: XP_002364923.1
-Organism (including strain): Toxoplasma gondii (strain ME49)
-Etiologic Risk Group (see link below): Risk group 2
-Disease Information (sort of like the Intro to your Mini Research Write up):
Toxoplasma gondii is a ubiquitous parasitic protozoan that causes the disease toxoplasmosis. Toxoplasmosis is a neurologic infection prevalent among people with acquired immunodeficiency syndrome or immunosuppressive cancer. However, humans with normal immunity can still contract the disease and certain symptoms, such as fever, malaise, and lymphadenopathy, will appear. Extreme cases include blindness, epilepsy, and even death [1]. T. gondii is estimated to infect half of the world's population, and it can virtually infect all warm-blooded animals [2]. Its economic impact is estimated to be $7.7 billion annually in the United States alone [3]. Infection among humans occurs mainly due to eating raw or uncooked meat, having contact with the parasite's oocysts via touching soil, or congenitally acquiring the disease through the placenta [1]. Currently, there is no licensed vaccine for humans [2], so much research still needs to be done.
-Link to TDR Targets page (if present): http://tdrtargets.org/targets/view?gene_id=259426
-Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.): NCBI, ToxoDB
-Essentiality of this protein: Pyruvate kinase is an important metabolic regulator in T. gondii that catalyzes the final step in glycolysis by converting phosphoenolpyruvate into pyruvate and producing ATP from ADP.
-Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): It is a multimer (4-mer).
-Complex of proteins?: Yes, 4 identical polypeptde strands arranged into 1 protein.
-Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): 0.2 (source)
-EC#: 2.7.1.40
-Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=2.7.1.40&Suchword=&reference=&UniProtAcc=&organism%5B%5D=Toxoplasma+gondii&show_tm=0
-Show screenshot of BRENDA enzyme mechanism schematic:
-Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Continuous spectrophotometric assay
-Link to Sigma (or other company) page for assay (see Sigma links below) or link (or citation) to paper that contains assay information: http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/pyruvkinase100mmimidazole.pdf
-Links to assay reagents (substrates) pages: http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/pyruvkinase100mmimidazole.pdf
-List cost and quantity of substrate reagents, supplier, and catalog #:
-100 mM imidazole HCl buffer, pH 7.2 at 30°C (from Sigma-Aldrich, product number: I-0250, price: $19.1/g)
-100 mM adenosine 5'-diphosphate (ADP) solution (from Sigma-Aldrich, product number: A-2754, price: $36.1/100mg)
-1000 mM magnesium chloride (MgCl2) solution (from Sigma-Aldrich, product number: M-0250, price: $55.5/500g)
-2500 mM potassium chloride (KCl) solution (from Sigma-Aldrich, product number: P-4504, price: $48.5/500g)
-155 mM phosphoenolpyruvate (PEP) solution (from Sigma-Aldrich, product number: P-3637, price: $65.7/500mg)
-13.1 mM nicotinamide adenine dinucleotide (NADH) reduced form solution (from Sigma-Aldrich, product number: N-8129, price: $27.3/50mg)
-L-Lactic dehydrogenase (LDH) enzyme solution (from Sigma-Aldrich, product number: L-2500, price: $41.3/5000 units)
-Pyruvate kinase (PK) enzyme solution (from Sigma-Aldrich, product number: P-7768, price: $35.5/kilounit)
Structure (PDB or Homology model)
-PDB # or closest PDB entry if using homology model: 3EOE
-For Homology Model option:
-Show pairwise alignment of your BLASTP search in NCBI against the PDB
-Query Coverage:
-Max % Identities:
-% Positives
-Chain used for homology:
-Current Inhibitors:
D-fructose-1-phosphate
D-glucose-1-phosphate
GTP
-Expression Information (has it been expressed in bacterial cells): Pyruvate kinase has been expressed in BL21(DE3) competent E. coli cells.
-Purification Method: Metal ion affinity with the enzyme's 6x His tag (source)
-Image of protein (PyMol with features delineated and shown separately):
-Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MHHHHHHSSGRENLYFQGIRMSQILEPRSEEDWTAHRTRIVCTMGPACWNVDTLVKMIDAGMNVCRLNFSHGDHETHART
VQNIQEAMKQRPEARLAILLDTKGPEIRTGFLKDHKPITLQQGATLKIVTDYNLIGDETTIACSYGALPQSVKPGNTILI
ADGSLSVKVVEVGSDYVITQAQNTATIGERKNMNLPNVKVQLPVIGEKDKHDILNFGIPMGCNFIAASFVQSADDVRYIR
GLLGPRGRHIRIIPKIENVEGLVNFDEILAEADGIMIARGDLGMEIPPEKVFLAQKMMIAKCNVVGKPVITATQMLESMI
KNPRPTRAEAADVANAVLDGTDCVMLSGETANGEFPVITVETMARICYEAETCVDYPALYRAMCLAVPPPISTQEAVARA
AVETAECVNAAIILALTETGQTARLIAKYRPMQPILALSASESTIKHLQVIRGVTTMQVPSFQGTDHVIRNAIVVAKERE
LVTEGESIVAVHGMKEEVAGSSNLLKVLTVE
-Length of your protein in Amino Acids: 511 amino acids
-Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 55.863 kDa
-Molar Extinction coefficient of your protein at 280 nm wavelength: 24410 L/mol.cm (assuming all Cys residues are reduced) and 25035 L/mol.cm (assuming all pairs of Cys residues form cystines)
-TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
-CDS Gene Sequence (paste as text only):
atgcatcatcatcatcatcatagcagcggccgcgaaaacctgtattttcagggcattcgc atgagccagattctggaaccgcgcagcgaagaagattggaccgcgcatcgcacccgcatt gtgtgcaccatgggcccggcgtgctggaacgtggataccctggtgaaaatgattgatgcg ggcatgaacgtgtgccgcctgaactttagccatggcgatcatgaaacccatgcgcgcacc gtgcagaacattcaggaagcgatgaaacagcgcccggaagcgcgcctggcgattctgctg gataccaaaggcccggaaattcgcaccggctttctgaaagatcataaaccgattaccctg cagcagggcgcgaccctgaaaattgtgaccgattataacctgattggcgatgaaaccacc attgcgtgcagctatggcgcgctgccgcagagcgtgaaaccgggcaacaccattctgatt gcggatggcagcctgagcgtgaaagtggtggaagtgggcagcgattatgtgattacccag gcgcagaacaccgcgaccattggcgaacgcaaaaacatgaacctgccgaacgtgaaagtg cagctgccggtgattggcgaaaaagataaacatgatattctgaactttggcattccgatg ggctgcaactttattgcggcgagctttgtgcagagcgcggatgatgtgcgctatattcgc ggcctgctgggcccgcgcggccgccatattcgcattattccgaaaattgaaaacgtggaa ggcctggtgaactttgatgaaattctggcggaagcggatggcattatgattgcgcgcggc gatctgggcatggaaattccgccggaaaaagtgtttctggcgcagaaaatgatgattgcg aaatgcaacgtggtgggcaaaccggtgattaccgcgacccagatgctggaaagcatgatt aaaaacccgcgcccgacccgcgcggaagcggcggatgtggcgaacgcggtgctggatggc accgattgcgtgatgctgagcggcgaaaccgcgaacggcgaatttccggtgattaccgtg gaaaccatggcgcgcatttgctatgaagcggaaacctgcgtggattatccggcgctgtat cgcgcgatgtgcctggcggtgccgccgccgattagcacccaggaagcggtggcgcgcgcg gcggtggaaaccgcggaatgcgtgaacgcggcgattattctggcgctgaccgaaaccggc cagaccgcgcgcctgattgcgaaatatcgcccgatgcagccgattctggcgctgagcgcg agcgaaagcaccattaaacatctgcaggtgattcgcggcgtgaccaccatgcaggtgccg agctttcagggcaccgatcatgtgattcgcaacgcgattgtggtggcgaaagaacgcgaa ctggtgaccgaaggcgaaagcattgtggcggtgcatggcatgaaagaagaagtggcgggc agcagcaacctgctgaaagtgctgaccgtggaa
-GC% Content for gene: 56.8%
-CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
ATGCACCACCATCACCATCATTCTTCTGGCCGCGAGAATTTATACTTCCAGGGTATTCGCATGTCTCAAATCCTGGAACCCCGTAGCGAAGAAGATTGGACCGCTCATCGCACACGCATCGTGTGTACGATGGG
ACCAGCGTGTTGGAACGTCGATACGCTGGTGAAAATGATTGACGCAGGTATGAATGTCTGCCGTTTAAATTTTTCACATGGTGATCACGAGACGCATGCGCGCACTGTGCAGAATATCCAAGAGGCTATGAAAC
AACGTCCCGAGGCCCGCCTTGCGATCTTATTGGATACGAAAGGGCCCGAAATCCGTACGGGGTTCCTGAAAGATCACAAACCAATCACCTTACAGCAAGGAGCAACGTTGAAAATTGTCACAGATTACAATTTA
ATTGGAGACGAGACAACCATCGCGTGTAGCTATGGGGCTTTGCCACAATCGGTTAAGCCGGGAAATACAATCTTAATTGCGGACGGCTCATTGAGCGTTAAGGTAGTGGAGGTAGGGTCCGATTATGTAATTAC
TCAAGCTCAAAACACTGCTACAATCGGAGAGCGTAAGAATATGAATCTGCCCAATGTTAAGGTACAACTGCCCGTCATTGGTGAAAAGGACAAGCACGACATTCTTAACTTTGGAATTCCAATGGGCTGTAATTT
TATTGCGGCAAGCTTCGTTCAGTCTGCTGACGACGTCCGTTACATCCGTGGACTGCTTGGTCCGCGTGGTCGCCATATCCGCATCATCCCCAAGATTGAAAATGTGGAAGGATTAGTGAACTTTGATGAGATCC
TTGCAGAGGCAGACGGCATTATGATCGCACGTGGTGACCTGGGGATGGAGATCCCCCCGGAGAAGGTCTTCTTGGCACAAAAAATGATGATCGCGAAATGTAATGTCGTGGGAAAGCCAGTCATCACGGCCA
CACAAATGTTGGAGTCCATGATCAAAAATCCCCGTCCGACTCGCGCGGAAGCGGCGGATGTAGCTAACGCGGTTCTGGATGGCACTGATTGCGTGATGCTGTCCGGCGAAACCGCAAACGGTGAGTTTCCT
GTCATCACTGTTGAGACTATGGCACGTATTTGTTATGAAGCGGAAACGTGTGTGGATTACCCTGCATTATACCGTGCGATGTGCTTAGCTGTACCCCCCCCGATCTCTACTCAAGAGGCGGTAGCTCGTGCCGC
AGTAGAGACGGCTGAATGCGTGAATGCCGCTATCATTTTGGCATTGACAGAAACGGGACAAACTGCCCGCTTAATCGCAAAGTACCGCCCCATGCAGCCCATTCTTGCCTTATCGGCTTCAGAGTCGACTATTA
AGCATCTTCAGGTAATTCGCGGAGTGACAACAATGCAGGTGCCTAGTTTCCAAGGTACGGATCACGTCATTCGTAACGCAATTGTTGTGGCTAAAGAGCGCGAGTTAGTAACAGAGGGCGAGTCCATTGTGGC
CGTTCACGGTATGAAAGAGGAAGTAGCGGGAAGCTCTAATTTATTGAAAGTTTTAACAGTAGAA
-GC% Content for gene (codon optimized): 49.1%
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
-References:
1. Jones, J.L.; Kruszon-Muran, D.; Wilson, M.; McQuillan, G.; Navin, T.; McAuley, J.B., Toxoplasma gondii Infection in the United States: Seroprevalence and Risk Factors. American Journal of Epidemiology 2001, 154, (4), 357-365.
2. Liu, Q.; Das Singla, L.; Zhou, H., Vaccines against Toxoplasma gondii: Status, challenges, and future directions. Human Vaccines and Immunotherapeutics 2012, 8, (9), 1305-1308.
3. Buzby, J.C.; Roberts, T., ERS updates US foodborne disease costs for seven pathogens. Food Review: The Magazine of Food 1996, 19, 20-25.